Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose [meTH-Seq]

Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation. Each experiment consists of two libraries, one reverse transcribed in low (restrictive) Mg2+ and one at permissive concentrations. Nm sites are identified through peaks, ie. positions where there are more reverse transcription stops in the restrictive than in the permissive condition. We include data for 41 such experiments across two growth conditions, and in various knockout backgrounds.

非编码RNA（non-coding RNAs）携带有数十种化学性质各异的修饰类型，而在信使RNA（messenger RNA，mRNA）中目前仅鉴定出其中少数几种。近期有研究发现部分转运RNA（transfer RNA，tRNA）修饰酶也可靶向作用于mRNA，这暗示mRNA或许存在大量尚未被发现的修饰类型。本研究通过交联免疫沉淀测序（crosslinking immunoprecipitation and sequencing，CLIP-seq）技术，证实酿酒酵母中保守的tRNA 2'-O-甲基转移酶Trm3、Trm7、Trm13与Trm44，以及核糖体RNA（ribosomal RNA，rRNA）2'-O-甲基转移酶Spb1，可与mRNA的特定位点相结合。本研究开发了双羟基甲基化测序（methylation at two prime hydroxyls，MeTH-seq）技术，可在转录组范围内以单核苷酸分辨率定位2'-O-甲基核糖（2′-O-methyl ribose，Nm）修饰，并在mRNA中发现了数千个潜在的Nm修饰位点。遗传学分析鉴定出Spb1甲基转移酶的数百个mRNA靶标，该酶可同时靶向mRNA与非编码RNA，实现受环境调控的修饰过程。本研究证实Nm是一种广泛存在且大概率具有保守性的mRNA修饰类型，并提供了用于研究其分布与调控机制的技术方法。每项实验均包含两个测序文库：一个是以低浓度（限制性条件）镁离子（Mg²+）进行反转录制备的文库，另一个则是以允许性浓度镁离子制备的文库。Nm修饰位点可通过峰形信号进行鉴定，即限制性条件下的反转录终止位点数量多于允许性条件的核苷酸位置。本数据集包含41组此类实验的数据，涵盖两种生长条件以及多种基因敲除背景。